Compositions and methods for antigen targeting to CD180

ABSTRACT

The present invention provides compositions of CD180 targeting molecules coupled to heterologous antigens, and their use in treating and/or limiting disease.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/245,459, filed Jan. 11, 2019, which is a continuation of U.S. patentapplication Ser. No. 14/426,635, filed Mar. 6, 2015, now granted U.S.Pat. No. 10,196,614 issued Feb. 5, 2019, which claims priority to U.S.national phase of International Application No. PCT/US2013/059898 filedon Sep. 16, 2013, and U.S. Provisional Application No. 61/702,368, filedSep. 18, 2012, all of which are incorporated by reference herein intheir entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under Grant Nos.A1052203 and A1044257, awarded by the National Institutes of Health. Thegovernment has certain rights in the invention

BACKGROUND

Current vaccination strategies require T cell help to produce IgG classantibodies, consequently they fail in patients with T cell deficiencies(DiGeorge Syndrome, AIDS) or defects in co-stimulation between B and Tcells (Hyper-IgM Syndrome from lack of CD40-CD40 ligand interaction).Antigen targeting is a method where an immunization material (antigen)is coupled to a monoclonal antibody that when injected both delivers theantigen and leads to a stimulatory signal to the antibody producing Bcell. Published antigen targeting strategies directed at CD40 or DEC-205on dendritic cells enhance IgG production but still require functional Tcells for this effect. Thus, improved compositions and methods areneeded.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides compositions,comprising:

(a) a CD180 targeting molecule; and

(b) a heterologous antigen attached to the CD 180 targeting molecule.

In various embodiments, the heterologous antigen is a polypeptide,carbohydrate, polysaccharide, or glycolipid antigen. In various furtherembodiments, the antigen is an allergen.

or a pathogen-specific antigen. In specific embodiments, thepathogen-specific antigen comprises an antigen selected from the groupconsisting of a West Nile virus antigen, a hepatitis virus antigen, anda dengue virus antigen. In another embodiment, the CD180 targetingmolecule is a CD180 antibody or antibody fragment. In a furtherembodiment, the CD180 targeting molecule is a CD180 antibody or antibodyfragment, and wherein the heterologous antigen is a polypeptide. Inanother embodiment, the composition further comprises an adjuvant, suchas toll-like receptor 7 (TLR7) agonists and TLR9 agonist.

In another aspect, the invention provides isolated nucleic acidsencoding the compositions of the invention where the antigen is apolypeptide. In a further aspect, the invention provides nucleic acidvector comprising the isolated nucleic acids of the invention. Inanother aspect, the invention provides recombinant host cells comprisingthe nucleic acid vectors of the invention. In a still further aspect,the invention provides pharmaceutical compositions, comprising acomposition according to any embodiment or combination of embodiments ofthe invention, and a pharmaceutically acceptable carrier.

In a further aspect, the invention provided methods for producingantibody, comprising culturing the recombinant host cells of theinvention under conditions suitable for expression of the nucleic-acidencoded antibody composition; and isolating the antibody compositionfrom the cultured cells.

In a still; further aspect, the invention provides methods for treatingor limiting development of a disorder, comprising administering to anindividual at risk of a disorder or having a disorder, an amounteffective to treat or limit development of the disorder of a compositionaccording to any embodiment or combination of embodiments of theinvention, or pharmaceutically acceptable salts thereof. In oneembodiment, the individual has a T-cell deficiency and/or a defect inco-stimulation between B cells and T cells. In another embodiment, theindividual is a neonate or is elderly. In further embodiments, theindividual has an allergy, a congenital or acquired immunodeficiency,has been exposed to an infectious agent, has one or more conditionsselected from the group consisting of ataxia-telangiectasia, hyper-IgMsyndrome, DiGeorge Syndrome, Wiscott-Aldrich Syndrome, Common VariableImmunodeficiency Syndromes, Polysaccharide response defects includingSelective Antibody Deficiency with Normal Immunoglobulins (SADNI), viralinfection, cancer, hepatitis, diabetes, and immunosuppression followingbone marrow or organ transplants or cytotoxic/myeloablative therapy; oris a pregnant female, asplenic, taking drugs that cause myelosuppressionor reduction in lymphocytes, receiving radiation therapy, and/or haschronic renal failure.

DESCRIPTION OF THE FIGURES

FIG. 1A-1C. Targeting to CD180 induces Ag-specific IgG production thatis partially T cellindependent. (A) WT mice were inoculated i.v. witheither 100 μg of NP-isotype control mAb (white bars) or graded doses ofNP-αCD180 mAb (10, 30 or 100 μg, light grey, medium grey and black barsrespectively); mice were pre-bled at d 0 (100 μg NP-αCD180 group only,hatched bars) or d 10 and total serum Ig (top) or anti-NP specific(bottom) IgM, IgG1 and IgG3 levels determined by ELISA. (B) WT, CD40 KO,or TCR KO mice were inoculated with 100 μg NP-αCD180 mAb (black) orNP-isotype mAb (white) or αCD180 (hatched), bled at d 10, and analyzedfor NP-specific IgM, IgG1 and IgG3 responses. (C) WT or CD40 KO micewere treated as in (B), bled at d 0 and d 10, and sera analyzed forlevels of NP-specific Abs (IgM and IgG subclasses). Data arerepresentative of 3 experiments (A), 4 experiments (B), and 3experiments (C) using 3 mice/group.

FIG. 2A-2C. CD180 targeting rapidly induces higher levels of Ag-specificIgG than Ag in alum. (A) WT or CD40 KO mice were inoculated i.v. witheither 100 μg NP-αCD180 (circles) or NP-isotype (upside down triangles)or i.p. with 100 μg NP-isotype in alum (triangles), bled at theindicated time points and serum analyzed for levels of NP-specific IgG.(B) WT mice were inoculated i.v. with either 100 μg OVA-αCD180 orOVA-isotype, or i.p. with 100 μg OVA-isotype in alum, bled at day 7 p.i.and serum analyzed for levels of OVA-specific IgG. (C) WT mice wereinoculated with 100 μg each of the indicated stimuli and bled on d 10and evaluated for levels of NP specific IgG (white columns) orOVA-specific IgG (black columns). Three mice/group, data representativeof 2 experiments (A), or 3 experiments (B, C).

FIG. 3A-3I. CD180 targeting induces affinity maturation, EF responses,germinal center formation and immunologic memory. (A) Sera from WT(black circles) or CD40 KO mice (open circles) immunized with 100 μgNP-αCD180 or 10 μg NP-αDCIR2 were analyzed for affinity to NP on days 5and 7 p.i. (B) WT (black circles) were inoculated with 100 μg NP-αCD180alone or with the indicated adjuvant (50 μg CpG-A, 50 μg CpG-B, 20 μgR848, or 4 μg LPS) then bled at d 7 and d 28; sera were analyzed foraffinity against NP. Controls included mice inoculated with NP iso,NP-iso+alum or NP-αDCIR2. (C) WT mice were inoculated with 50 μgNP-αCD180 alone or with the indicated adjuvants as in (B) and bled at d7, 14, 21 and 28; sera were analyzed for levels of NP-specific IgM(left) or IgG (right) Abs. A representative experiment of threeexperiments each for A, B, and C is shown. (D-F) 2×105 NP-binding Bcells from Ly5.1+ B1-8hi mice were adoptively transferred to Ly5.2+WTrecipients on d −1. On d 0 the mice were inoculated with 100 μg ofeither NP-αCD180 (black) or NP-isotype (white) and spleens harvested ateither d 4 or d 7 for flow cytometric analysis. The number ofNP-specific B220+ B cells (D) and NP-specific PNA+GL7+GC B cells (E) perspleen were determined by sequential gating on Ly5.1+NP-binding B cells.(F) The number of B220loCD138+AFCs per spleen are plotted. Datarepresentative of two experiments for D-F. (G) 8 μM frozen spleensections from mice immunized with NP-CGG+alum or NP-αCD180 either 4 or 7days previously were stained with anti-B220-eFluor450, PNA-FITC, andNP-PE. Data are representative of multiple sections analyzed from 2-3mice per time point. Scale bars represent 100 μM. (H) Mean number ofPNA+GCs per follicle from mice in (G) (day 7 time point). Each dotrepresents one animal from which 4 individual sections were analyzed.Data in G and H depict a representative experiment of 2 independentexperiments. (I) WT and CD40 KO mice were primed as indicated, restedfor 10 wks, then given a secondary challenge i.p. with Ag (20 μg) orPBS. Spleens were harvested d 4 post-boost and analyzed for NP-specificAFC by ELISPOT. The combined results from two independent experimentsusing 3 mice/group are shown. Each dot represents an individual animal.Statistical values compare groups to mice primed with NP-αCD180 andchallenged with NP-isotype. * p<0.05; ** p<0.01; *** p<0.001.

FIG. 4A-4B. Ag-specific B cells are efficiently activated in vivo bycombined signaling through BCR and CD180. (A, B) Groups of B1-8hi mice(9 mice/group) were inoculated with either 100 μg NP-isotype orNP-αCD180 and spleens were harvested 24 h later (A and B) or 48 and 72 hlater (data not shown). The NP-specific B cells (6-10%) weredistinguished from total CD19+ B cells by staining with NP-APC. Fourgroups of CD19+ B cells were then analyzed ex vivo for their expressionof CD69, CD86, MHC class II, and TACI: unstimulated B cells (NP-B cellsfrom NP-isotype-treated mice, gray in (A); B-cell receptor(BCR)-stimulated B cells (NP+ B cells from NP-isotype treated mice);CD180-stimulated B cells (NP-B cells from NP-αCD180 treated mice); and Bcells stimulated through both the BCR and CD180 stimulated (NP+ B cellsfrom NP-αCD180 treated mice). (A) Histograms show CD86 and TACIexpression on B220+ B cells stimulated as indicated 24 hpost-immunization. (B) Mean fluorescent intensities at 24 hpost-immunization are plotted for the indicated surface markers relativeto unstimulated control B cells (value 1.0). Similar data were obtainedat 48 and 72 h post-immunization. A representative experiment of threeexperiments is shown.

FIG. 5A-5C. CD180 Ag-targeting responses require expression on B cellsand not on non-B cells. (A) Schematic of adoptive transfers. 10×10⁶ Bcells purified from either WT or CD180 KO mice were transferred to μMTor CD180 KO recipients as indicated. 24 h following transfer, mice wereinoculated with 100 μg of NP-isotype or NP-αCD180 and bled 10 d later.(B) NP-specific IgG responses at d 10 of groups shown in (A) afterinoculation with 100 μg NP-αCD180 (black) or NP-isotype (white) analysisby ELISA. Three mice/group; representative of two experiments. * p<0.05;*** p<0.001. (C) WT mice were inoculated with 100 μg OVA-αCD180 orOVA-isotype and spleens harvested 16 h later. B cells and DCs werepurified by negative selection, then seeded at indicated ratios intoculture with CFSE-labeled purified OT-II T cells at the indicatedratios; CFSE dilution of CD4+Vα2 TCR+ cells was assessed following 72 hin culture. Co-cultures performed in triplicate, representative of twoindependent experiments for C.

FIG. 6A-6C. MHC class II is required for Ag targeting to CD180 but notBAFF-R, IFNα/β, IL-4 or OX40L. (A, B) WT C57BL/6 mice and the indicatedKO mice (A) or BALB/c mice (B) were inoculated with 100 μg NP-αCD180 orNP-isotype, and bled at d 10; levels of NP-specific IgG Abs weredetermined by ELISA. Three mice/group; representative of two experimentsfor both A and B. * p<0.05; ** p<0.01; *** p<0.001 as determined byone-way ANOVA with Bonferonni post-tests by comparing to WT controls.(C) Groups of WT mice were immunized with 100 μg NP-αCD180 or NP-isotypeand sacrificed on days 1 and 3 p.i. for analysis of splenic B cellsubsets. Flow cytometry plots show gating strategy used to enumerate FO(B220+CD23hiCD21int), MZ (B220+CD23loCD21hiCD93−) and T1/T2 transitional(B220+CD23loCD21loCD93+) B cells (day 1 group shown). Bar graphs depicttotal number of cells in the spleen (mean+/−SEM) of each subset. Numberon graphs indicate fold increase in cell subsets fromNP-αCD180-immunized mice compared to isotype controls. Data are from oneexperiment using 3-4 mice/group/time point. * p<0.05; ** p<0.01; ***p<0.001 as determined by one-way ANOVA with Bonferonni post-tests.

FIG. 7A-7B. Graph showing that combination of an OVA-αCD180 conjugateand a TLR7 agonist provide synergistic induction of (A) IgG and (B) IgMantibody responses.

FIG. 8A-8B. Graph showing that mice inoculated with anti-CD180 to whichpurified recombinant West Nile virus (WNV) envelope (E) protein has beenattached (WNVE) develop both (A) WNVE specific IgG Abs and (B)neutralizing Abs to WNVE.

FIG. 9 . Graph showing that the WNVE-αCD180 conjugate induces aneutralizing antibody response that is enhanced by the addition of anadjuvant to TLR7 (R848) or TLR9 (CpGB).

DETAILED DESCRIPTION OF THE INVENTION

All references cited are herein incorporated by reference in theirentirety. Within this application, unless otherwise stated, thetechniques utilized may be found in any of several well-known referencessuch as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press), Gene Expression Technology(Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. AcademicPress, San Diego, Calif.), “Guide to Protein Purification” in Methods inEnzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCRProtocols: A Guide to Methods and Applications (Innis, et al. 1990.Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual ofBasic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York,N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J.Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998Catalog (Ambion, Austin, Tex.).

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. “And” as usedherein is interchangeably used with “or” unless expressly statedotherwise.

All embodiments of any aspect of the invention can be used incombination, unless the context clearly dictates otherwise.

In a first aspect, the present invention provides compositions,comprising:

(a) a CD180 targeting molecule; and

(b) a heterologous antigen attached to the CD 180 targeting molecule.

The compositions of the invention can be used, for example, in methodstargeting antigen to CD180, a surface protein expressed on B cells,macrophages, and dendritic cells, that to produce antigen-specific IgGin the absence of T cell costimulation (such as CD40 deficiency) or thecomplete absence of T cells (such as TCR β/δ deficiency).

As disclosed herein, the inventors have surprisingly discovered that thecompositions of the invention induce rapid activation of antigen(Ag)-specific B cells, leading to significant antigen-specific IgG andIgM production within 7 days. Remarkably, a single injection of Ag-CD180antibody (αCD180) without any additional adjuvant also led to thedevelopment of both antibody (Ab) affinity maturation and immunologicmemory.

The CD180 targeting molecule may be any molecule that binds directly toCD180 present in the surface of B cells, macrophages, or dendriticcells. In various non-limiting embodiments, the CD180 targeting moleculemay be a polypeptide (such as a peptide mimetic, antibody, etc.),nucleic acid (such as an aptamer), carbohydrate, organic molecule, etc.In a particular embodiment, the CD180 targeting molecule comprises apolypeptide; in one non-limiting embodiment, the polypeptide is anantibody or antibody fragment. As used herein, “antibody” includesreference to an immunoglobulin molecule immunologically reactive withhuman CD180 (preferably selective for CD180), and includes monoclonalantibodies. Various isotypes of antibodies exist, for example IgG1,IgG2, IgG3, IgG4, and other Ig, e.g., IgM, IgA, IgE isotypes. The termalso includes genetically engineered forms such as chimeric antibodies(e.g., humanized murine antibodies) and heteroconjugate antibodies(e.g., bispecific antibodies), fully humanized antibodies, and humanantibodies. As used throughout the application, the term “antibody”includes fragments with antigen-binding capability (e.g., Fab′, F(ab′)₂,Fab, Fv and rIgG. See also, Pierce Catalog and Handbook, 1994-1995(Pierce Chemical Co., Rockford, Ill.). See also, e.g., Kuby, J.,Immunology, 3^(rd) Ed., W.H. Freeman & Co., New York (1998). The termalso refers to recombinant single chain Fv fragments (scFv). The termantibody also includes bivalent or bispecific molecules, diabodies,triabodies, and tetrabodies. Bivalent and bispecific molecules aredescribed in, e.g., Kostelny et al. (1992) J Immunol 148:1547, Pack andPluckthun (1992) Biochemistry 31:1579, Hollinger et al., 1993, supra,Gruber et al. (1994) J Immunol:5368, Zhu et al. (1997) Protein Sci6:781, Hu et al. (1996) Cancer Res. 56:3055, Adams et al. (1993) CancerRes. 53:4026, and McCartney, et al. (1995) Protein Eng. 8:301. Variousantigen binding domain-fusion proteins are also disclosed, e.g., in USpatent application Nos. 2003/0118592 and 2003/0133939, and areencompassed within the term “antibody” as used in this application.

An antibody immunologically reactive with human CD180 can be generatedby recombinant methods such as selection of libraries of recombinantantibodies in phage or similar vectors, see, e.g., Huse et al., Science246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989); andVaughan et al., Nature Biotech. 14:309-314 (1996), or by immunizing ananimal with the antigen or with DNA encoding the antigen.

In one embodiment, the antibody comprises or consists mAb G28-8, whichis commercially available from a number of sources, (Thermo Scientific,Sigma Aldrich, etc.) or a F(ab′)2 fragment of mAb G28-8. In a particularembodiment, the antibody comprises a human or animal CD180 bindingdomain linked to an immunoglobulin constant region (Fc) domain that hasimpaired binding to human or animal Fc receptor FcγRIIb and/or to humanor animal complement proteins. For example, the antibody or fragmentthereof may be one such as is described in US patent applicationpublication number 20120020965, incorporated by reference herein in itsentirety. For example, the antibody may comprise or consist of the aminoacid sequence of SEQ ID NO:2, a scFv-Fc molecule constructed from thecloned variable regions of mAb G28-8. The Fc domain of the recombinantmolecules is an altered human IgG1 Fc domain with three amino acidchanges (P238S, P331S, K322S) that reduce the binding of the molecule toFc-receptors and C1q.

The compositions comprise a “heterologous” antigen, in that the antigenis not naturally present in the CD180 targeting molecule; thus, thecompositions comprise a conjugate of the CD180 targeting molecule andthe heterologous antigen. The CD180 targeting molecule and theheterologous antigen can be conjugated in any suitable manner, includingcovalently and non-covalently; any manner that irreversibly links theheterologous Ag to the anti-CD180 should work. The specific manner ofconjugation most appropriate for a specific composition will depend onthe components utilized, and can be determined by those of skill in theart. In some embodiments, the targeting molecule and the antigen can beconjugated via one or more linker moieties. In embodiments where boththe targeting molecule and the antigen are polypeptides, thecompositions can be recombinantly expressed as a chimera. Fornon-recombinantly expressed embodiments, any suitable conjugationtechnique can be used, including but not limited to engineered disulfidelinkages, formalin/glutaraldehyde crosslinking, selective linking (suchas is disclosed in the examples that follow), streptavidin/biotin basedconjugation, bispecifics with CD180 and tag (FLAG, His, etc.) bindingability could be generated and mixed with tagged Ag or have directaffinity for the Ag, etc.

Since the compositions can be used in methods targeting antigen to CD180to produce antigen-specific IgG in the absence of T cell costimulationor the complete absence of T cells, it will be clear to those of skillin the art that any suitable antigen can be used in the compositions.The heterologous antigen may be of any compound type, including but notlimited to polypeptide, nucleic acid, lipid, polysaccharide, glycolipid,etc. In one embodiment the heterologous antigen comprise a polypeptide.In other embodiments, the heterologous antigen comprises apolysaccharide or a glycolipid antigen.

In one embodiment, the heterologous antigen comprises or consists of apathogen-specific antigen; the antigen may be from any pathogen ofinterest in a given situation. In non-limiting embodiments, thepathogen-specific antigens include antigens from hepatitis (A, B, C, E,etc.) virus, human papillomavirus, herpes simplex viruses,cytomegalovirus, Epstein-Barr virus, influenza virus, parainfluenzavirus, enterovirus, measles virus, mumps virus, polio virus, rabiesvirus, human immunodeficiency virus, respiratory syncytial virus,Rotavirus, rubella virus, varicella zoster virus, Ebola virus,cytomegalovirus, Marburg virus, norovirus, variola virus, any Flavivusincluding but not limited to West Nile virus, yellow fever virus, denguevirus, tick-borne encephalitis virus, and Japanese encephalitis virus;human immunodeficiency virus (HIV), Bacillus anthraces, Bordetallapertusis, Chlamydia trachomatis, Clostridium tetani, Clastridiumdifficile, Corynebacterium diptheriae, Coxiella burnetii, Escherichiacoli, Haemophilus influenza, Helicobacter pylori, Leishmania donovani,L. tropica and L. braziliensis, Mycobacterium tuberculosis,Mycobacterium leprae, Neisseria meningitis, Plasmodium falciparum, P.ovale, P. malariae and P. vivax, Pseudomonas aeruginosa, Salmonellatyphi, Schistosoma hematobium, S. mansoni, Streptococcus pneumoniae(group A and B), Staphylococcus aureus, Toxoplasma gondii, Trypanosomabrucei, T cruzi and Vibrio cholerae.

In one non-limiting example, the heterologus antigen is an antigen fromWest Nile virus (WNV), including but not limited to WNV envelope proteinE with an amino acid sequence similar to or identical to those described(T. J Chambers et al., J General Virology 79:2375-2380, 1998) orantigenic fragments thereof or a WNV nonstructural (NS) protein such asNS2a, NS2b, NS3, NS4a, NS4b or NS5. As shown in the examples thatfollow, mice inoculated with a CD180 targeting molecule to whichpurified recombinant West Nile virus (WNV) envelope (E) protein has beenattached (WNVE) develop both WNVE specific IgG Abs and neutralizing Absto WNVE, and that mice immunized with WNVE-CD180 conjugates areprotected from death induced by intracranial challenge with WNV. Thesedata are merely exemplary of how the compositions of the invention canbe used to induce rapid activation of antigen (Ag)-specific B cells andT cells against any antigen conjugated to the CD180 targeting molecule,leading to significant IgG and IgM production, Ab affinity maturation,and immunologic memory.

In another embodiment, the heterologous antigen is an antigen fromanother flavivirus such as hepatitis C virus (HCV) or dengue virus(DENV). The antigen from HCV is an antigen including but not limited toHCV capsid protein C, envelope proteins E1 and E2, and nonstructuralproteins NS2, NS3, NS4a, NS4b, NS5a and NS5b (C. Wychowski et al. J.Virol 67:1385, 1993) or antigen fragments thereof. In the case of theHCV E2 protein, the sequence may be e.g., as described in Q L Choo etal. (PNAS 88:2451-2458, 1991). The antigen from DENV is an antigenincluding but not limited to envelope (E) protein or antigenic fragmentsthereof from one or more of the four DENV serotypes.

In another embodiments, the heterologous antigen is a hepatitis B virussurface antigen (HBsAg) with amino acid sequence such as that describedby P Charnay et al. (Nucleic Acids Res 7:335-346, 1979) or D. L.Peterson et al (J. Biol. Chem 257:10414-10420, 1982) or HBsAg variants(J. N Zuckerman and A. J Zuckerman, Antiviral Res 60:75-78, 2003).

In another embodiment, the heterologous antigen comprises or consists ofan allergen (i.e.: any substance that can cause an allergy), includingbut not limited to allergens found in animal products (fur and dander;wool, dust mite excretion, etc.); drugs (penicillin, sulfonamides,salicylates, etc.); food (celery, corn, eggs/albumin, fruit,milk/lactose, seafood, sesame, legumes (beans, peas, peanuts, soybeans,etc); soy, tree nuts (pecans, almonds, etc.)); insect stings (bee stingvenom, wasp sting venom, mosquito stings, etc.); mold spores, and plantpollens (grass, weeds, ragweed, trees, etc.)

In a further embodiment, the heterologous antigen comprises or consistsof other types of disease-related antigens against which it would bebeneficial to generate an immune response against, including but notlimited to antigens expressed in or on the surface of tumors/tumor cells(including but not limited to p53 (colorectal cancer), alphafetoprotein(germ cell tumors; hepatocellular carcinoma), carcinoembryonic antigen(bowel cancers), CA-125 (ovarian cancer), human epidermal growth factorreceptor-2 (HER-2, breast cancer), MUC-1 (breast cancer), NY-ESO-1(esophageal cancer, non-small-cell lung cancer), epithelial tumorantigen (breast cancer), tyrosinase (malignant melanoma),Disialoganglioside (GD2, neuroblastoma), melanoma-associated antigengene-1 (MAGE-1 (malignant melanoma)), and beta amyloid (for Alzheimer'sand other amyloid-based diseases), etc.

In another embodiment, the composition of any embodiment or combinationof embodiments of the invention further comprises an adjuvant. While theexamples below demonstrate that adjuvant is not required to induce rapidactivation of Ag-specific B cells, leading to significant IgG and IgMproduction within 7 days, development of both Ab affinity maturation,and immunologic memory, the examples further show that addition ofadjuvant to the compositions can result in additional enhancement of theimmune response when the compositions are used in the methods of theinvention. Any suitable adjuvant can be used, including but not limitedto inorganic compounds (aluminum hydroxide, aluminum phosphate, calciumphosphate hydroxide, beryllium, etc.), mineral oil, detergents,cytokines, toll-like receptor agonists, Freund's complete adjuvant,Freund's incomplete adjuvant, squalene, etc. In a preferred embodiment,the adjuvant comprises or consists of a toll-like receptor (TLR)agonist, and more preferably a TLR7 (including but not limited tosynthetic small molecule imidazoquinolines, such as imiquimod orresiquimod (R848); CAS number: 144875-48-9; available from InvivoGen)and/or a TLR9 agonist ((including but not limited to Type A, B, or C CpGoligonucleotides; available from InvivoGen). As shown in the examplesthat follow, use of the compositions of the invention in combinationwith a TLR7 agonist and/or a TLR9 agonist provide a synergisticinduction of the IgG and the IgM responses, enhances the neutralizing Abresponse against the WNVE antigen after WNVE-αCD180 conjugateadministration, and enhances activation and expansion of the cytotoxic Tcell response.

The adjuvant may be present in the composition as an unlinked component,or may be linked to the antigen-CD180 targeting molecule conjugate,depending on the adjuvant and conjugate used. In various non-limitingembodiments, the adjuvant in the composition may comprise flagellins(TLR5 agonists) or nucleic acid agonists of TLR7 and TLR9 that can besynthesized with modified bases allowing linkage to the anti-CD180 (suchas via llylamine linkages).

In another embodiment, the compositions of the invention can be modifiedto extend half-life, such as by attaching at least one molecule to thecomposition for extending serum half-life, including but not limited toa polyethlyene glycol (PEG) group, serum albumin, transferrin,transferrin receptor or the transferrin-binding portion thereof, orcombinations thereof. As used herein, the word “attached” refers to acovalently or noncovalently conjugated substance. The conjugation may beby genetic engineering or by chemical means.

The compositions of the present invention may be stored in any suitablebuffer.

In a second aspect, the present invention provides isolated nucleicacids encoding the composition of any embodiment of the first aspect ofthe invention where the targeting molecule and the antigen arepolypeptides. The isolated nucleic acid sequence may comprise RNA orDNA. Such isolated nucleic acid sequences may comprise additionalsequences useful for promoting expression and/or purification of theencoded protein, including but not limited to polyA sequences, modifiedKozak sequences, and sequences encoding epitope tags, export signals,and secretory signals, nuclear localization signals, and plasma membranelocalization signals.

In a third aspect, the present invention provides nucleic acid vectorscomprising the isolated nucleic acid of the second aspect of theinvention. “Recombinant expression vector” includes vectors thatoperatively link a nucleic acid coding region or gene to any promotercapable of effecting expression of the gene product. The promotersequence used to drive expression of the disclosed nucleic acidsequences in a mammalian system may be constitutive (driven by any of avariety of promoters, including but not limited to, CMV, SV40, RSV,actin, EF) or inducible (driven by any of a number of induciblepromoters including, but not limited to, tetracycline, ecdysone,steroid-responsive). The construction of expression vectors for use intransfecting prokaryotic cells is also well known in the art, and thuscan be accomplished via standard techniques. (See, for example,Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer andExpression Protocols, pp. 109-128, ed. E. J. Murray, The Humana PressInc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin,Tex.). The expression vector must be replicable in the host organismseither as an episome or by integration into host chromosomal DNA. In apreferred embodiment, the expression vector comprises a plasmid.However, the invention is intended to include other expression vectorsthat serve equivalent functions, such as viral vectors.

In a fourth aspect, the present invention provides recombinant hostcells comprising the nucleic acid vector of the third aspect of theinvention. The host cells can be either prokaryotic or eukaryotic. Thecells can be transiently or stably transfected. Such transfection ofexpression vectors into prokaryotic and eukaryotic cells (including butnot limited to Chinese hamster ovary (CHO) cells) can be accomplishedvia any technique known in the art, including but not limited tostandard bacterial transformations, calcium phosphate co-precipitation,electroporation, or liposome mediated-, DEAE dextran mediated-,polycationic mediated-, or viral mediated transfection. (See, forexample, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manualof Basic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. NewYork, N.Y.).

The recombinant host cells can be used, for example in methods forproducing antibody (when the targeting molecule is an antibody),comprising:

(a) culturing the recombinant host cell of the invention underconditions suitable for expression of the nucleic-acid encoded antibodycomposition; and

(b) isolating the antibody composition from the cultured cells.

Suitable conditions for expression of the nucleic-acid encoded antibodycomposition can be determined by those of skill in the art based on theteachings herein, the specific host cells and vectors used, and thegeneral knowledge of those of skill in the art.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under expressed or notexpressed at all. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,in a form not normally found in nature. In this manner, operably linkageof different sequences is achieved. Thus an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes disclosed herein. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes disclosed herein.

In a fifth aspect, the present invention provides pharmaceuticalcompositions, comprising:

(a) the composition of embodiment or combination of embodimentsdisclosed herein; and

(b) a pharmaceutically acceptable carrier.

In this embodiment, the compositions of the invention are present in apharmaceutical formulation. In this embodiment, the compositions arecombined with a pharmaceutically acceptable carrier. Suitable acidswhich are capable of forming such salts include inorganic acids such ashydrochloric acid, hydrobromic acid, perchloric acid, nitric acid,thiocyanic acid, sulfuric acid, phosphoric acid and the like; andorganic acids such as formic acid, acetic acid, propionic acid, glycolicacid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinicacid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid,naphthalene sulfonic acid, sulfanilic acid and the like. Suitable basescapable of forming such salts include inorganic bases such as sodiumhydroxide, ammonium hydroxide, potassium hydroxide and the like; andorganic bases such as mono-, di- and tri-alkyl and aryl amines (e.g.,triethylamine, diisopropyl amine, methyl amine, dimethyl amine and thelike) and optionally substituted ethanol-amines (e.g., ethanolamine,diethanolamine and the like).

The pharmaceutical composition may comprise in addition to thecomposition of the invention (a) a lyoprotectant; (b) a surfactant; (c)a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) apreservative and/or (g) a buffer. In some embodiments, the buffer in thepharmaceutical composition is a Tris buffer, a histidine buffer, aphosphate buffer, a citrate buffer or an acetate buffer. Thepharmaceutical composition may also include a lyoprotectant, e.g.sucrose, sorbitol or trehalose. In certain embodiments, thepharmaceutical composition includes a preservative e.g. benzalkoniumchloride, benzethonium, chlorohexidine, phenol, m-cresol, benzylalcohol, methylparaben, propylparaben, chlorobutanol, o-cresol,p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoicacid, and various mixtures thereof. In other embodiments, thepharmaceutical composition includes a bulking agent, like glycine. Inyet other embodiments, the pharmaceutical composition includes asurfactant e.g., polysorbate-20, polysorbate-40, polysorbate-60,polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitanmonolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitanmonooleate, sorbitan trilaurate, sorbitan tristearate, sorbitantrioleaste, or a combination thereof. The pharmaceutical composition mayalso include a tonicity adjusting agent, e.g., a compound that rendersthe formulation substantially isotonic or isoosmotic with human blood.Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine,methionine, mannitol, dextrose, inositol, sodium chloride, arginine andarginine hydrochloride. In other embodiments, the pharmaceuticalcomposition additionally includes a stabilizer, e.g., a molecule which,when combined with a protein of interest substantially prevents orreduces chemical and/or physical instability of the protein of interestin lyophilized or liquid form. Exemplary stabilizers include sucrose,sorbitol, glycine, inositol, sodium chloride, methionine, arginine, andarginine hydrochloride.

The pharmaceutical compositions of the invention may be made up in anysuitable formulation, preferably in formulations suitable foradministration by injection. Such pharmaceutical compositions can beused, for example, in methods of use as vaccines, prophylactics, ortherapeutics.

The pharmaceutical compositions may contain any other components asdeemed appropriate for a given use, such as additional therapeutics orvaccine components. In one embodiment, the pharmaceutical compositionsfurther comprise toll-like receptor agonists including but not limitedto Ribi, a TLR7 agonist (including but not limited to R848) and/or aTLR9 agonist ((including but not limited to Type A, B, or C CpGoligonucleotides).

In a sixth aspect, the present invention provides methods for treatingor limiting development of a disorder, comprising administering to anindividual at risk of a disorder an amount effective to treat or limitdevelopment of the disorder of the composition or pharmaceuticalcomposition, or a pharmaceutical salt thereof, of any embodiment orcombination of embodiments of the present invention. In one embodiment,the compositions are used prophylactically as vaccines to limitinfectious disease/severity of infectious disease, such as inindividuals that have not been exposed to an infectious agent but are atrisk of such exposure. In other embodiments, the compositions can beused therapeutically to treat people exposed to or chronically infectedwith a pathogen. In a further embodiment, the compositions are used ascancer vaccines, to treat an individual with a tumor. As will beunderstood by those of skill in the art, the specificantigen/composition to be used will depend on the specific disorder tobe treating or limited.

Suitable acids which are capable of forming pharmaceutically acceptablesalts include inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid,phosphoric acid and the like; and organic acids such as formic acid,acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid,oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid,anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilicacid and the like. Suitable bases capable of forming pharmaceuticallyacceptable salts include inorganic bases such as sodium hydroxide,ammonium hydroxide, potassium hydroxide and the like; and organic basessuch as mono-, di- and tri-alkyl and aryl amines (e.g., triethylamine,diisopropyl amine, methyl amine, dimethyl amine and the like) andoptionally substituted ethanol-amines (e.g., ethanolamine,diethanolamine and the like).

The methods of the invention target antigen to CD180, a surface proteinexpressed on B cells, macrophages, and dendritic cells, that to produceantigen-specific IgG in the absence of T cell costimulation (such asCD40 deficiency) or the complete absence of T cells (such as TCR β/δdeficiency). Thus, the methods can be used in any therapeutic orprophylactic treatment for which a suitable antigen is available. Asshown in the examples that follow, induction of antigen specific IgG andIgM by anti-CD180 conjugates is rapid (within 7 days as opposed to 14+days for most conventional immunization systems) and requires noadditional inflammation-inducing adjuvant. Remarkably, a singleinjection of Ag-CD180 antibody (αCD180) without any additional adjuvantalso led to the development of both antibody (Ab) affinity maturationand immunologic memory. Furthermore, as shown in the examples thatfollow, mice inoculated with a CD180 targeting molecule to whichpurified recombinant West Nile virus (WNV) envelope (E) protein has beenattached (WNVE) develop both WNVE specific IgG Abs and neutralizing Absto WNVE, and that mice immunized with WNVE-CD180 conjugates areprotected from death induced by intracranial challenge with WNV. Thesedata are merely exemplary of how the methods of the present inventioncan be used to treat or limit development of a disease.

Thus, the approach of targeting antigen to CD180 results in bothsignificant advantages compared to traditional (e.g. IM antigen in alum)immunization approaches and allows for vaccination of previouslyuntreatable populations. This approach also finds use, for example, forneonates, the elderly, and the immunodeficient, both in specificallytargeting cellular populations enriched in underdeveloped or otherwisedeficient immune systems and by improving responses to antigens thatrequire linked recognition (carbohydrate epitopes, etc.).

As used herein, “treat” or “treating” means accomplishing one or more ofthe following in an individual that already has a disorder or hasalready been exposed to a disorder-causing substance/pathogen: (a)reducing the severity of the disorder; (b) limiting or preventingdevelopment of symptoms characteristic of the disorder(s) being treated(ex: immune deficiencies in cancer patients or other patients)undergoing chemotherapy and/or radiation therapy); (c) inhibitingworsening of symptoms characteristic of the disorder(s) being treated;(d) limiting or preventing recurrence of the disorder(s) in patientsthat have previously had the disorder(s); and (e) limiting or preventingrecurrence of symptoms in patients that were previously symptomatic forthe disorder(s).

As used herein, “limiting” or “limiting development of” meansaccomplishing one or more of the following in an individual that doesnot have the disorder to be limited: (a) preventing the disorder; (b)reducing the severity of the disorder; and (c) limiting or preventingdevelopment of symptoms characteristic of the disorder.

As used herein, an “amount effective” refers to an amount of thecomposition that is effective for treating and/or limiting the relevantdisorder.

While the examples below demonstrate that adjuvant is not required toinduce rapid activation of Ag-specific B cells, leading to significantIgG and IgM production within 7 days, development of both Ab affinitymaturation, and immunologic memory, the examples further show thataddition of adjuvant to the compositions can result in additionalenhancement of the immune response when the compositions are used in themethods of the invention. Thus, in a further embodiment, the methods mayfurther comprise administering an adjuvant to the individual. Anysuitable adjuvant can be used, including but not limited to inorganiccompounds (aluminum hydroxide, aluminum phosphate, calcium phosphatehydroxide, beryllium, etc.), mineral oil, detergents, cytokines,toll-like receptor agonists, Freund's complete adjuvant, Freund'sincomplete adjuvant, squalene, etc. In a preferred embodiment, theadjuvant comprises or consists of adjuvant comprises or consists of atoll-like receptor (TLR) agonist, and more preferably a TLR7 (includingbut not limited to synthetic small molecule imidazoquinolines, such asimiquimod or resiquimod (R848); CAS number: 144875-48-9; available fromInvivoGen) and/or a TLR9 agonist ((including but not limited to Type A,B, or C CpG oligonucleotides; available from InvivoGen). As shown in theexamples that follow, use of the compositions of the invention incombination with a TLR7 agonist and/or a TLR9 agonist provide asynergistic induction of the IgG and the IgM responses, enhances theneutralizing Ab response against the WNVE antigen after WNVE-αCD180conjugate administration, and enhances activation and expansion of a CD4T cell response or a cytotoxic T cell response.

The individual may be any suitable individual, including but not limitedto mammals. Preferably the individual is a human. In one embodiment, theindividual has a T-cell deficiency and/or a defect in co-stimulationbetween B cells and T cells, or is immuno-compromised by chronicinfections or from acute or chronic taking of immunosuppressive drugsfor treatment of autoimmune diseases, or other inflammatory disease. Inanother embodiment, the individual is a neonate or is elderly (i.e.: atleast 65 years old).

In various other embodiments, the individual has an allergy, has acongenital or acquired immunodeficiency, or has been exposed to aninfectious agent.

In various further embodiments, the individual suffers from one or moreof ataxia-telangiectasia, hyper-IgM syndrome, DiGeorge Syndrome,Wiscott-Aldrich Syndrome, Common Variable Immunodeficiency Syndromes,Polysaccharide response defects including Selective Antibody Deficiencywith Normal Immunoglobulins (SADNI), viral infection, cancer, hepatitis,diabetes, and immunosuppression following bone marrow or organtransplants or cytotoxic/myeloablative therapy. or is a pregnant female,asplenic, taking drugs that cause myelosuppression or reduction inlymphocytes (including but not limited to corticosteroids, cyclosporine,amphotericin B, chloramphenicol, various cancer chemotherapeutics, goldcompounds, methotrexate, etc.), receiving radiation therapy, and/or haschronic renal failure.

As will be understood by those of skill in the art, the methods can beused to treat or limit development of any disorder mediated by apathogen or allergen for which an appropriate antigen can be provided inthe composition, including but not limited to hepatitis (A, B, C, E,etc.) virus, human papillomavirus, herpes simplex viruses,cytomegalovirus, Epstein-Barr virus, influenza virus, parainfluenzavirus, enterovirus, measles virus, mumps virus, polio virus, rabiesvirus, human immunodeficiency virus, respiratory syncytial virus,Rotavirus, rubella virus, varicella zoster virus, Ebola virus,cytomegalovirus, Marburg virus, norovirus, variola virus, any Flavivusincluding but not limited to West Nile virus, yellow fever virus, denguevirus, tick-borne encephalitis virus, and Japanese encephalitis virus;human immunodeficiency virus (HIV), Bacillus anthraces, Bordetallapertusis, Chlamydia trachomatis, Clostridium tetani, Clastridiumdifficile, Corynebacterium diptheriae, Coxiella burnetii, Escherichiacoli, Haemophilus influenza, Helicobacter pylori, Leishmania donovani,L. tropica and L. braziliensis, Mycobacterium tuberculosis,Mycobacterium leprae, Neisseria meningitis, Plasmodium falciparum, P.ovale, P. malariae and P. vivax, Pseudomonas aeruginosa, Salmonellatyphi, Schistosoma hematobium, S. mansoni, Streptococcus pneumoniae(group A and B), Staphylococcus aureus, Toxoplasma gondii, Trypanosomabrucei, T cruzi and Vibrio cholerae; allergens found in animal products(fur and dander; wool, dust mite excretion, etc.); drugs (penicillin,sulfonamides, salicylates, etc.); food (celery, corn, eggs/albumin,fruit, milk/lactose, seafood, sesame, legumes (beans, peas, peanuts,soybeans, etc); soy, tree nuts (pecans, almonds, etc.)); insect stings(bee sting venom, wasp sting venom, mosquito stings, etc.); mold spores,plant pollens (grass, weeds, ragweed, trees, etc; antigens expressed inor on the surface of tumors/tumor cells (including but not limited top53 (colorectal cancer), alphafetoprotein (germ cell tumors;hepatocellular carcinoma), carcinoembryonic antigen (bowel cancers),CA-125 (ovarian cancer), human epidermal growth factor receptor-2(HER-2, breast cancer), MUC-1 (breast cancer), NY-ESO-1 (esophagealcancer, non-small-cell lung cancer), epithelial tumor antigen (breastcancer), tyrosinase (malignant melanoma), Disialoganglioside (GD2,neuroblastoma), melanoma-associated antigen gene-1 (MAGE-1 (malignantmelanoma)), and beta amyloid (for Alzheimer's and other amyloid-baseddiseases), etc.

Thus, the methods can be used, for example, to treat or limitdevelopment of any disorders associated with the antigens listed above(i.e. relevant viral and bacterial infections, chickenpox, cervicalcancer, genital warts, gastroenteritis, smallpox, anthrax, whoopingcough, tetanus, diphtheria, meningitis, viral encephalitis, malaria,diarrhea, pneumonia, acquired immune deficiency syndrome, tuberculosis,cholera, typhoid fever, cancer, Alzheimer's disease, etc.).

In one non-limiting embodiment, the method is used to treat or limitdevelopment of disease caused by West Nile virus (WNV) infection. Inthis embodiment, the heterologous antigen is an antigen from West Nilevirus (WNV), including but not limited to including but not limited toWNV envelope protein E with an amino acid sequence similar to oridentical to those described (T. J Chambers et al., J General Virology79:2375-2380, 1998) or antigenic fragments thereof or a WNVnonstructural (NS) protein such as NS2a, NS2b, NS3, NS4a, NS4b or NS5.or antigenic fragments thereof. As shown in the examples that follow,mice inoculated with a CD180 targeting molecule to which purifiedrecombinant West Nile virus (WNV) envelope (E) protein has been attached(WNVE) develop both WNVE specific IgG Abs and neutralizing Abs to WNVE,and that mice immunized with WNVE-CD180 conjugates are protected fromdeath induced by intracranial challenge with WNV. These data are merelyexemplary of how the methods of the invention can be used to treat orlimit development of a disorder for which an appropriate antigen isavailable for generating an immune response against.

Symptoms characteristic of disease caused by WNV include, but are notlimited to headache, fever, rash, neuroinvasive disease, encephalitis,meningitis, meningioencephalitis, and poliomyelitis. Thus, the methodsof the invention may serve, for example, to therapeutically treat orlimit development of these symptoms and/or to limit WNV replication insomeone exposed to WNV prior to the methods, or to prophylacticallylimit development of these symptoms and/or to limit WNV replication insomeone exposed top WNV only after the methods of the invention.

In another embodiment, the method is used to treat or limit developmentof disease caused by another flavivirus such as hepatitis C virus (HCV)or dengue virus (DENV). In this embodiment, the heterologous antigenfrom HCV can be an antigen including but not limited to HCV capsidprotein C, envelope proteins E1 and E2, and nonstructural proteins NS2,NS3, NS4a, NS4b, NS5a and NS5b (C. Wychowski et al. J. Virol 67:1385,1993) or antigen fragments thereof. In the case of the HCV E2 protein,the sequence may be e.g., as described in QL Choo et al. (PNAS88:2451-2458, 1991). The antigen from DENV can be an antigen includingbut not limited to envelope (E) protein or antigenic fragments thereoffrom one or more of the four DENV serotypes.

In another embodiment, the method is used to treat or limit developmentof disease caused by a hepatitis B virus, where the antigen may be, forexample, surface antigen (HBsAg) with amino acid sequence such as thatdescribed by P Charnay et al. (Nucleic Acids Res 7:335-346, 1979) or D.L. Peterson et al (J. Biol. Chem 257:10414-10420, 1982) or HBsAgvariants (J. N Zuckerman and A. J Zuckerman, Antiviral Res 60:75-78,2003).

The compositions are typically formulated as a pharmaceuticalcomposition, such as those disclosed above, and can be administered viaany suitable route, including orally, parentally, by inhalation spray,rectally, or topically in dosage unit formulations containingconventional pharmaceutically acceptable carriers, adjuvants, andvehicles. Preferably, the compositions are administered parenterally.The term parenteral as used herein includes, subcutaneous, intravenous,intra-arterial, intramuscular, intrasternal, intratendinous,intraspinal, intracranial, intrathoracic, infusion techniques orintraperitoneally. Dosage regimens can be adjusted to provide theoptimum desired response (e.g., a therapeutic or prophylactic response).A suitable dosage range may, for instance, be 0.1 ug/kg-100 mg/kg bodyweight; alternatively, it may be 0.5 ug/kg to 50 mg/kg; 1 ug/kg to 25mg/kg, or 5 ug/kg to 10 mg/kg body weight. The compositions can bedelivered in a single bolus, or may be administered more than once(e.g., 2, 3, 4, 5, or more times) as determined by an attendingphysician.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words “comprise,” “comprising,” and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to.” Words using the singular or pluralnumber also include the plural or singular number, respectively.Additionally, the words “herein,” “above,” and “below” and words ofsimilar import, when used in this application, shall refer to thisapplication as a whole and not to any particular portions of thisapplication. As used herein, the singular forms “a”, “an” and “the”include plural referents unless the context clearly dictates otherwise.“And” as used herein is interchangeably used with “or” unless expresslystated otherwise.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments.

All of the references cited herein are incorporated by reference.Aspects of the disclosure can be modified, if necessary, to employ thecompositions, functions and concepts of the above references andapplication to provide yet further embodiments of the disclosure. Theseand other changes can be made to the disclosure in light of the detaileddescription.

The following description provides specific details for a thoroughunderstanding of, and enabling description for, embodiments of thedisclosure. However, one skilled in the art will understand that thedisclosure may be practiced without these details. In other instances,well-known structures and functions have not been shown or described indetail to avoid unnecessarily obscuring the description of theembodiments of the disclosure.

Example: Targeting Antigens to CD180 Rapidly Induces Antigen-SpecificIgG, Affinity Maturation and Immunologic Memory

Summary

Here we report that targeting antigen (Ag) to a receptor expressed onboth B cells and dendritic cells (DCs), the TLR orphan receptor CD180,in the absence of adjuvant rapidly induced IgG responses that werestronger than those induced by Ag in alum. Ag conjugated to anti-CD180(Ag-αCD180) induced affinity maturation and antibody (Ab) responses thatwere partially T cell-independent, as Ag-specific IgG were generated inCD40- and T cell-deficient mice. After pre-immunization with Ag-αCD180and boosting with soluble Ag both WT and CD40 KO mice rapidly producedAg-specific IgG forming cells, demonstrating that Ag-anti-CD180 inducesimmunologic memory. The potent adjuvant effect of Ag-αCD180 required Agto be coupled to anti-CD180 and the responsive B cells to express bothCD180 and an Ag-specific B cell receptor (BCR). Surprisingly, CD180Ag-targeting also induced IgG Abs in BAFF-R KO mice lacking mature Bcells and in mice deficient in interferon-signaling. Targeting Ag toCD180 is thus useful for therapeutic vaccination and for vaccinating theimmune-compromised.

Results

Targeting Antigen to CD180 Rapidly Induces Strong Ag-Specific IgGResponses

We examined whether Ag coupled to anti-CD180 could induce Ag-specificIgG responses in normal and immunodeficient mice. We first conjugatedthe hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) to anti-CD180(NP-αCD180) or to a non-binding rat IgG2a isotype control (NP-isotype)mAb and administered them in graded doses i.v. to WT mice. Doses rangingfrom 10- to 100 μg of NP-αCD180 induced significant NP-specific IgGresponses in a dose dependent manner (FIG. 1 A, bottom) with little orno polyclonal Ig production compared to unimmunized mice (pre-bleed for100 μg NP-αCD180 group) or mice injected with 100 μg NP-isotype (FIG. 1A, top). Anti-NP Abs were not observed in mice immunized with anti-CD180mAb alone (FIG. 1 B); therefore, the Ag-specific Ab response toNP-αCD180 was due to targeting of Ag rather than from a product ofpolyclonal Ig production. Strong Ag-specific Ab response to NP-αCD180were also induced when conjugates were inoculated i.p. (data not shown);in subsequent studies we inoculated mice via the i.v. route.

Targeting Ag to CD180 also induced Ag-specific IgM and IgG production inboth CD40 KO mice and T cell deficient (TCRβ/δ KO) mice (FIG. 1 B).Ag-specific IgM levels were similar in the WT and immunodeficient mice,but Ag-specific IgG levels were significantly lower in both CD40 KO andTCRβ/δ KO mice. Despite the overall reduction in Ag-specific IgG inimmunodeficient mice, the broad IgG subclass distribution was maintainedand similar to that in WT mice (FIG. 1 C). In addition to Ag-specificIgG, NP-αCD180 also induced Ag-specific IgA Abs, but not IgE Abs (datanot shown). We conclude that targeting Ag to CD180 induces both TD andTI IgG antibody responses.

CD180 Targeting Rapidly Induces Higher Levels of Ag-Specific IgG than AgInoculated in Alum

We next determined the kinetics of Ag-specific IgG production followingNP-αCD180 inoculation. We immunized WT or CD40 KO mice i.v. with eitherNP-αCD180 or NP-isotype, or i.p. with the NP-isotype Ag precipitated inalum. In WT mice, NP-αCD180 induced a rapid anti-NP IgG response thatpeaked 10 d p.i. as compared to Ag in alum, which peaked on d 21. Miceinoculated with NP-isotype alone did not produce more than 2 μg/mL ofanti-NP Ab at any time point (FIG. 2 A, left). As expected, CD40 KO miceimmunized with Ag in alum did not make an NP-specific IgG response;however, they did develop a significant and continually increasingamount of NP-specific IgG after CD180 targeting (FIG. 2 A, right).

Targeting to CD180 Induces Anti Protein IgG Responses and RequiresCovalently Linked Ag

We next determined whether the strong Ab response to NP-αCD180 was alsoinduced when we targeted protein antigens to CD180. We coupled whole OVAto anti-CD180 (OVA-αCD180) and isotype mAb (OVA-isotype) and immunizedWT mice i.v. with one of these antigens or i.p. with OVA-isotype in alum(FIG. 2 B). As with NP-αCD180, OVA-αCD180 induced a strong Ag-specificIgG response with concentrations of nearly 2 mg/mL of IgG anti-OVA atday 14 p.i.

Anti-CD180 alone can stimulate B cells and thus has the potential toconvert B cells into efficient antigen presenting cells (APCs) so theycould present Ag even if it were administered in an unlinked fashion. Totest this possibility, we inoculated mice with two different Ags withonly one Ag coupled to αCD180: NP-αCD180+soluble OVA orOVA-αCD180+soluble NP-isotype. As expected, mice inoculated with onlyNP-isotype in alum or OVA in alum produced IgG only against NP or OVA,respectively (FIG. 2 C). Mice inoculated with NP or OVA coupled toanti-CD180 together with soluble OVA or soluble NP-isotype only made Absagainst the Ag coupled to anti-CD180 and not to the soluble, unlinkedAg. We conclude that during Ag targeting to CD180, only B cells specificfor the Ag attached to anti-CD180 are driven to produce Ab.

CD180 Targeting Induces Affinity Maturation, EF Responses, GerminalCenter (GC) Formation and Immunologic Memory

To assess whether CD180 targeting alone or with the addition ofadjuvants could induce affinity maturation of Abs, we inoculatedNP-αCD180 alone (50 μg i.v.) or co-administered with TLR-based adjuvantsincluding CpG A or CpG B (TLR9), R848 (TLR7) or LPS (TLR4) and obtainedsera 5, 7, or 28 d thereafter. To measure changes in relative affinity,we measured the relative binding of antisera to BSA with low levels ofNP bound (NP2) vs. to BSA with higher levels of NP bound (NP20). For anegative control we immunized mice with NP-αDCIR2, which we hadpreviously shown does not induce affinity maturation (Chappell et al.,2012). Following immunization with NP-αCD180, Ab affinity increased fromdays 5-7 (FIG. 3 A) to levels significantly above the affinity afterimmunization with NP-αDCIR2, and this difference was still evident on d28 (FIG. 3 B, bottom). Immunization of mice with unconjugated αCD180plus TLR agonists had no effect on anti-NP Ab levels (data not shown).The addition of adjuvants along with NP-αCD180 did not change Abaffinity at d 7 (FIG. 3 B, top) even though it increased NP-specific IgMand IgG production 4- to 7-fold (FIG. 3 C). By d 28 after immunizationthe addition of a CpG adjuvant significantly increased affinity whilethe other adjuvants did not (FIG. 3 B, bottom). Unlike in WT mice, theaffinity of the IgG Abs induced in CD40 KO mice did not increase abovethe levels of the negative controls (FIG. 3 A).

To follow expansion and differentiation of Ag-specific B cells afterimmunization with NP-αCD180, we adoptively transferred splenocytescontaining NP-specific B cells from Ly5.1+ B1-8hi mice (Shih et al.,2002) into Ly5.2+WT hosts. Spleens were harvested at d 4 or d 7following inoculation with NP-αCD180 or NP-isotype control and analyzedby flow cytometry using sequential gating for B220+, Ly5.1+, and NP-APCbinding. NP-isotype-treated mice showed no expansion of Ag-specificB220hi B cells, while NP-αCD180-treated mice showed an approximately20-fold expansion at both time points (FIG. 3 D). This expansionincluded GL7+PNA+GC B cells, which increased in number by d 7 (FIG. 3E). By d 7 the NP-αCD180-treated mice also had significant numbers ofCD138+ antibody forming cells (AFCs) in the spleen (FIG. 3 F). Theseresults suggested that NP-αCD180 induces both EF Ab responses and GCformation. Indeed, by d 4 the spleens of NP-αCD180-treated mice hadlarge numbers of Ag-specific B cells in EF sites and by d 7 PNA+GCs wereevident (FIG. 3 G). By comparison, GCs induced by NP-αCD180 weregenerally smaller (FIG. 3 G) and present in fewer numbers (FIG. 3 H)than those induced by NP-CGG plus alum.

The presence of GCs in NP-αCD180-treated mice suggested that theAg-specific B cell expansion induced by Ag-αCD180 leads to thedevelopment of immunologic memory, which is characterized by the abilityto rapidly generate Ag-specific AFCs in response to soluble Agre-challenge. To test this, we immunized groups of WT and CD40 KO micewith NP-conjugated mAbs as above or with NP-chicken gamma globulin (CGG)in alum as a positive control. After 10 weeks mice were boosted withmatched soluble Ag (NP-isotype or NP-CGG) or with PBS as a negativecontrol. On d 4 post-boost spleens were harvested and the number ofIgG-producing AFCs was assessed using an NP-specific IgG ELISPOT assay.As expected, WT mice primed with NP-CGG in alum produced significantnumbers of IgG-producing AFCs when boosted with soluble Ag (FIG. 3 I,left). In contrast to mice primed with NP-isotype, NP-αCD180-primed micecontained low but detectable numbers of Ag-specific long-lived plasmacells (LLPCs) in the spleen 10 weeks following immunization (0.0 vs.5.7+/−0.8 SEM per 10⁶ splenocytes). Levels of Ag-specific AFCs increasedapproximately 10-fold upon re-challenge with soluble Ag (56+/−8.7 SEMper 10⁶ splenocytes). The number of AFCs generated in mice primed withNP-αCD180 in the absence of adjuvant was roughly one third that ofNP-CGG in alum primed mice. This result is in accord with smaller GCinduction by NP-αCD180 compared to NP-CGG plus alum (FIGS. 3 G and 3 H).However, the spot size was three times as large as spots from miceprimed with NP-CGG (data not shown), suggesting that the amount ofNP-specific IgG produced per AFC was greater. Addition of the adjuvantR848 during the primary immunization with NP-αCD180 neither increasedthe spot number nor spot size upon Ag re-challenge compared to miceprimed with NP-αCD180 alone.

Surprisingly, following re-challenge with soluble Ag, we also detectedsome NP-specific IgG-secreting AFCs in CD40 KO mice primed withNP-αCD180 (FIG. 3 I, right). While the number of NP-specificIgG-secreting AFCs in CD40 KO mice was roughly 1/15th the number in WTmice, this number was significantly higher than in PBS-boosted CD40 KOmice or CD40 KO mice primed with Ag in alum. Thus, CD180 targetingeffectively primes for immunologic memory in WT mice, and to a lesserextent even in the absence of CD40.

Ag-Specific B Cells are Efficiently Activated by Linking BCR and CD 180Stimuli

The fact that specific Ag must be linked to anti-CD180 in order toinduce IgG Ab responses (FIG. 2 C) suggested that both BCR and CD180ligation on the same cell are required for specific Ab to be produced.To test this possibility, first we compared the activation of B cellsfollowing stimulation in vivo through either the BCR, CD180, or throughboth receptors. We used B1-8hi mice that contain NP-specific B cells(Shih et al., 2002); groups of these mice were injected with either 100μg NP-αCD180 or with NP-isotype and spleens harvested 24 hrs later. TheNP-specific B cells (6-10%) were distinguished from total CD19+ B cellsby staining with NP-APC. Four groups of CD19+ B cells were then analyzedex vivo for their expression of CD69, CD86, MHC class II, and thereceptor, transmembrane activator and calcium-modulator and cyclophilinligand interactor (TACI): unstimulated B cells (NP-B cells fromNP-isotype-treated mice), BCR-stimulated B cells (NP+ B cells fromNP-isotype treated mice), CD180-stimulated B cells (NP-B cells fromNP-αCD180 treated mice), and B cells stimulated through both the BCR andCD180 stimulated (NP+ B cells from NP-αCD180 treated mice). Compared tounstimulated B cells, B cells stimulated via either Ag or αCD180 hadincreased expression of CD86 and TACI (FIG. 4 A). However, over a seriesof experiments, the levels of CD69, CD86, and TACI were significantlyhigher on B cells stimulated through both the BCR and CD180 at 24 h(FIGS. 4 A and B) and later time points (data not shown). Thus, thecombination of BCR and CD180 signaling in vivo appears to be moreeffective at activating B cells than either signal alone.

B Cell Expression of CD 180 is Necessary and Sufficient for Ag-αCD180Driven Ab Responses

The data in FIG. 4 suggest that the powerful adjuvant effect ofAg-αCD180 may be mediated by the combination of BCR and CD180 signalingof B cells. However, since CD180 is expressed on both B cells and non-Bcells, Ab responses induced by CD180 targeting may be mediated by eitherdelivery of both Ag-mediated BCR signaling together with CD180 signalsto Ag-specific B cells and/or CD180 delivery and signaling to non-Bcells which then in turn stimulate Ag-specific B cell and T cellresponses. To distinguish these possibilities we carried out adoptivetransfers to establish mice that express CD180 only on B cells, only onnon-B cells, or on both B cells and non-B cells (FIG. 5 A). B celldeficient μMT mice were inoculated with CD180 KO B cells to create micewhere CD180 was expressed only on non-B cells. These mice failed to makeAg-specific IgG after inoculation with NP-αCD180 (FIG. 5 B),demonstrating that CD180 expression on B cells is necessary to generatean Ab response after CD180 targeting. CD180 KO recipients, into whichpurified CD180+ B cells were transferred, expressed CD180 only on Bcells and not on non-B cells. Following immunization with NP-αCD180,these mice produced high levels of Ag-specific IgG. These data show thatCD180 expression on B cells is sufficient for CD180-based targeting. Bcell deficient (μMT) mice into which CD180+ B cells were transferred sothat CD180 was expressed on both B cells and non-B cells made somewhatmore NP-specific IgG than mice not expressing CD180 on non-B cells (FIG.5 B). This suggests that CD180 expression on non-B cells such as DCs,while neither sufficient nor essential for Ag targeting, influences theextent of IgG production.

When anti-CD180 mAb is inoculated i.v. into mice, it binds toCD180+CD19+ B cells and to other CD180+ cells in the spleen includingCD11c+ DCs and F4/80+ macrophages, but not to CD3+ T cells, which do notexpress CD180 (data not shown). To determine which APCs were mosteffective at priming T cells following targeting to CD180, WT mice wereinoculated with either OVA-isotype or OVA-αCD180; 16 h later B cells andDCs were purified by negative selection and co-cultured withCFSE-labeled OVA-specific OT-II CD4 T cells or OT-I CD8 T cells. After72 h the levels of CFSE in the OVA-specific T cells were measured byflow cytometry (FIG. 5 C). OVA-αCD180 targeted B cells, unlike B cellsfrom OVA-isotype treated control mice, clearly induced proliferation ofAg-specific CD4 T cells. However, OVA-αCD180-targeted DCs were much moreeffective on a per cell basis at stimulating OT-II proliferation.OVA-αCD180 targeted B cells, unlike OVA-αCD180 targeted DCs, failed toinduce any proliferation of OVA-specific OT-I CD8 T cells, consistentwith the poor cross-presentation of Ag by B cells compared to DCs. Thus,while DCs are not required for the Ag-specific Ab responses induced byAg-αCD180, they may function to stimulate Ag-specific CD4 helper T cellsrequired for optimal IgG production.

IL4, IFN-α/β Signaling and Mature B Cells are not Required forAg-Targeting to CD 180

Type I interferon (IFN) has been shown to act directly on B cells andpromote Ab responses (LeBon et al., 2005, Fink et al., 2006) Thus, wecompared IgG responses of type 1 IFN α/β receptor (IFNα/βR) KO and WTmice after inoculating NP-αCD180; abrogating signaling through theIFNα/βR if anything increased anti-NP IgG production, suggesting thattype 1 IFNs may normally restrain Ab responses induced via CD180. Micedeficient in MHC class II (MHC II KO) after immunization with NP-αCD180had a more severe reduction in anti-NP IgG production than either CD40or TCR KO mice (FIG. 6 A). The reason for this is not clear as B celllevels are normal in MHC II KO mice, and MHC II B cells respond normallyto TI Ags (Markowitz et al., 1993). Another T cell-dependent B cellactivator, αIgD, requires the action of IL-4 (Finkelman et al., 1986),so we compared Ab responses of WT, IL4 KO and OX40L KO mice inoculatedwith NP-αCD180. After CD180 targeting both IL-4 KO and OX40L KO miceproduced anti-NP IgG at levels similar to WT controls (FIG. 6 A, B),demonstrating that the IL-4-Th2 pathway is not required during CD180targeting.

In order to assess a possible role for the cytokine BAFF in CD180targeting, we immunized BAFF-R KO mice, which have a near complete blockin mature B cell development (Sasaki et al., 2004). To our surpriseBAFF-R KO mice produced normal levels of NP-specific IgG Abs followingtargeting to CD180 (FIG. 6 A). This suggests that while CD180+ B cellsare required for Ag-αCD180 targeting, mature B cells may not benecessary. Indeed, we observed significant increases in both FO(B220+CD23hi CD21int) and T1/T2 (B220+CD2310 CD2110 CD93+) transitionalB cells on days 1 and 3 following immunization with NP-αCD180 comparedto NPisotype-injected animals (FIG. 6 C). In contrast, B cells with a MZphenotype (B220+CD2310 CD21hi CD93−) showed a marked decrease followingNP-αCD180 administration. These results demonstrate that NP-αCD180expands some but not all splenic B cell subsets. Since BAFF can bind toTACI and BCMA as well as BAFF-R (Rickert et al., 2011), it remainspossible that signaling through TACI or BCMA contributes toAg-αCD180-driven IgG responses.

DISCUSSION

Collectively, our data indicate that targeting Ags to CD180 inducesrapid activation of Ag-specific B cells, leading to significant IgGproduction within 7 days. Remarkably, a single injection of Ag-αCD180without any additional adjuvant also led to the development of both Abaffinity maturation and immunologic memory (FIG. 3 ). Furthermore, whileseverely impaired, Ag-specific IgG production and responses to secondaryimmunizations were retained in CD40 KO mice (FIG. 3 H), even though CD40KO mice did not make Ag-specific IgG or develop memory Ab producingcells in response to Ag in alum, as reported previously (Kawabe et al.,1994). The Ab responses induced required the Ags to be attached toanti-CD180 and could be induced to both haptens and protein Ags.

Why is this mode of immunization so effective in rapidly raising IgG Abresponses? Several lines of evidence suggest that it is the combinationof simultaneous signaling of Ag-αCD180 through both the BCR and CD180 onB cells that promotes the rapid Ab responses. First, effective inductionof IgG by Ag-αCD180 required CD180 to be expressed on B cells and not onother cells. Second, while Ab responses to linked Ag occurred with bothNP-αCD180 and OVA-αCD180, there was little or no response to soluble Agsco-administered at the same time. Third, B cells activated in vivo bystimulating the Ag receptor and CD180 together expressed higher levelsof activation markers than B cells triggered by either stimulus alone(FIG. 4 ). Indeed, the greater induction of CD86 expression afterco-ligation of the BCR and CD180 may well be a feature of targeting Agto CD180, as CD86 is necessary for IgG responses to non-adjuvanted Ag(Borriello et al., 1997). The TACI receptor was also induced to higherlevels after CD180 targeting, and TACI plays a role in class switchingand IgG production (He et al., 2010).

Ag targeting to CD180, while requiring B cells, does not appear torequire mature B cells: BAFF-R KO mice mainly have transitional 1 (T1) Bcells; they have a fivefold reduction in transitional 2 B cells and arealmost completely deficient of mature follicular and marginal zone Bcells (Sasaki et al., 2004). Nevertheless, inoculation of Ag-αCD180 intoBAFF-R KO mice produced as much Ag-specific IgG as in WT mice. Thissuggests that T1 B cells are a major target for Ag-anti-CD180. AlthoughT1 B cells readily apoptose following BCR stimulation alone, they do notdie when signaled via BCR and a second signal (Kovesdi et al., 2004), T1B cells also constitutively express activation-induced deaminase (AID)(Ueda et al, 2007, Han et al., 2007, Kuraoka et al., 2009) and canrapidly produce large quantities of IgG and undergo somatic mutationwhen triggered with a combination of BCR and TLR stimuli (Mao et al.,2004, Ueda et al., 2007, Han et al., 2007, Capolunghi et al., 2008,Aranburu et al., 2010, Kuraoka et al., 2011). Thus, AID+T1 B cellssignaled through both the BCR and CD180 may rapidly switch and matureinto IgG-producing plasma cells.

Our results indicate that Ag-αCD180 targeting generates long-livedplasma cells and switched memory B cells in both WT and CD40 KO mice.First, the t1/2 of Ag-specific IgG in immunized WT mice wasapproximately 38 days (based on the kinetics in FIG. 2 A), whilecatabolism of a discrete burst of IgG from a short-lived AFC responsewould have a t1/2 of 21 days. Additionally, Ag-specific IgG levels inCD40 KO mice continue to rise over time. Both of these results requirecontinual IgG production to slow or offset the constant elimination IgG,implying that some Ab-producing cells are retained. Second, Ag-specificGL7+PNA+GC B cells were evident by d 7 after immunization withNP-αCD180. This GC phenotype suggests memory B cell precursors werebeing generated. Third, both WT and CD40 KO mice had significantly moreAFCs following Ag boost than mice primed with Ag-isotype or mice notboosted (FIG. 3 I). Many studies have implicated CD40 signals in theinduction of memory B cells by TD or TI-2 Ag (Taylor et al., 2012, Kajiet al., 2012), and indeed a much stronger memory response was induced byNP-αCD180 in WT mice than in CD40 KO mice. However, NP-αCD180 clearlyinduced some CD40-independent B cell memory. TI Ags can induce Tcell-independent GC-independent memory B cell responses (Zhang et al.,1988, Weller et al., 2001, Defrance et al., 2011, Berkowska et al.,2012). Thus, in addition to the generation of strong EF responses,stimulation through CD180, when combined with BCR signaling, may be anovel pathway of T-independent memory B cell differentiation.

Although CD40 KO and TCR-deficient mice still can make IgG after CD180targeting, the amount of Ag-specific IgG is only about 10% of that in WTmice. Thus, T cells clearly are required for most of the IgG response.Since CD180 is expressed on both B cells and DCs and internalizesfollowing ligation by mAb (data not shown), it was likely that CD180targeting could deliver Ag both to Ag-specific B cells as well as to DCsthat don't bind Ag. Indeed, this was the case: DCs targeted in vivo viaαCD180 were more efficient than targeted B cells in stimulating CD4 Tcell proliferation. Although Ab responses induced by αCD180 onlyrequired CD180 expression on B cells, it appears that DC-mediated T cellpriming helped promote a greater response to CD180 targeting in WT micethan if Ag were solely directed to B cells (FIG. 5 B).

The combined Ag targeting/adjuvant method described here can be used forhuman vaccines. Most vaccines do not induce protective immunity in allindividuals, and most vaccines do not induce lasting immunity.Furthermore, vaccination of immuno-compromised individuals requiresspecial considerations and approaches (Rappuoli et al., 2011, Miller andRathore, 2012). Targeting to CD180 induces IgG responses and someimmunologic memory even in CD40 KO mice, and, remarkably, induces highlevels of IgG Abs even in mature B cell-deficient BAFF-R KO mice andIFN-signaling-deficient IFNα/βR KO mice. Thus, a CD180-based vaccineplatform can be used for immunizing immuno-compromised people includingthe elderly. In addition, most vaccine strategies require more than oneinjection in order to produce sufficient circulating levels ofprotective Abs. Single dose vaccines provide a number of advantages(Bowick and McAuley, 2011, Levine, 2011) and one injection of Agattached to anti-CD180 induces a rapid and strong IgG response. Thus, asingle inoculation of a CD180-based vaccine can produce protectivehumoral immunity and be a particularly attractive approach fortherapeutic vaccination shortly after an exposure to a pathogen.

Materials and Methods

Mice

C57BL/6, CD40 KO, OT-I OVA-specific CD8+ TCR transgenic, OT-IIOVA-specific CD4+ TCR transgenic, B cell-deficient (μMT), and Tcell-deficient (TCRβ/δ KO) mice were purchased from Jackson Laboratory(Bar Harbor, Me.). All strains were on the C57BL/6 background unlessotherwise noted. CD180 KO, MHC II KO, and IFNα/βR KO mice were giftsfrom S. Skerrett, P. Fink and K. Murali-Krishna, respectively(University of Washington, Seattle, Wash.). OX40L KO mice were a giftfrom A. H. Sharpe (Harvard University, Cambridge, Mass.). BAFF-R KO micewere a gift from K. Rajewsky (Harvard Medical School, Boston, Mass.).B6.SJL-B1-8hi knockin Ly5.1 mice were a gift from M. Nussenzweig(Rockefeller University, New York, N.Y.). IL-4 KO mice on the BALB/cbackground were a gift from S. Ziegler (Benaroya Research Institute,Seattle, Wash.), and WT control BALB/c mice were purchased from theJackson Laboratory. All mice were sex- and age-matched and used at sixto ten weeks of age. The University of Washington Institutional AnimalCare and Use Committee approved all animal work.

Cell Preparation and Adoptive Transfers

Total splenocytes were processed by mechanical disruption anderythrocytes were depleted by Gey's lysis. For adoptive transferexperiments in FIG. 3 , splenocytes from B1-8hi IgH transgenic mice werelabeled with PE-conjugated NP and anti-B220-FITC to determine thefrequency of Ag-specific B cells by flow cytometry. Total splenocytescontaining 2×105 NP-binding B cells were transferred i.v. to individualB6 recipients 24 h prior to immunization. For experiments in FIGS. 5 Aand B, splenic B cells from WT or CD180-deficient mice were isolated bythree rounds of negative selection enrichment (STEMCELL Technologies,Vancouver, BC, Canada). 10×106 purified B cells of appropriate genotypewere transferred i.v. to recipients as indicated 24h prior toimmunization. For experiments in FIG. 5C, CD4 and CD8 T cells from OTIIand OT-I TCR transgenic mice, respectively, and DCs or B cells fromimmunized C57BL/6 mice were isolated by three rounds of negativeselection enrichment using the appropriate kit (STEMCELL Technologies,Vancouver, BC, Canada). Purities for all cell enrichments were >99% asdetermined by flow cytometry for CD19 (B cells), CD4 or CD8 (T cells),or CD11c (DCs). Frequencies of OT-I and OT-II T cells were determinedwithin the CD3+ T cell population by staining for Vα2 and used todetermine final numbers for cell culture.

In Vitro CFSE Proliferation Assay

5×10⁴ B cells or DCs from immunized mice were enriched as describedabove and co-cultured with titrating numbers of CFSE-labeled Vα2+OT-I orOT-II T cells in 96-well round bottom plates for 3 days at 37 C, 5% CO2as previously described (Chaplin et al., 2011). CFSE (Invitrogen)labeling was performed as previously described (Chaplin et al., 2011).

ELISA and ELISPOT Assays

For ELISA assays, polystyrene plates were coated with either 2 μg/mLanti-mouse IgG (H+L) (Jackson ImmunoResearch, West Grove, Pa.) for totalIg, 20 μg/mL NiP-BSA (Biosearch Technologies, Novato, Calif.) forNP-specific Ab, or 20 μg/mL OVA (Sigma-Aldrich). Affinity determinationswere performed as described previously (Herzenberg et al., 1980,Chappell et al., 2012), using custom NiP2- and NiP20-BSA prepared byconjugation to the succinimidyl ester of NiP (Biosearch Technologies)according to manufacturer instructions. Detection and analyses wereperformed as previously described (Chaplin et al., 2011). ELISPOT assayswere performed as previously described (Goins et al., 2010). Spot numberand size were quantified using a CTL-ImmunoSpot™ S5 Core AnalyzerELISPOT™ reader with ImmunoSpot™ Academic V5.0 software (CellularTechnology Ltd., Shaker Heights, Ohio).

Flow Cytometry

Flow cytometry analyses were performed on a FACSCanto™ (BectonDickinson, Franklin Lakes, N.J.). A minimum of 30,000 cells of the finalgated population was used for all analyses. Data analyses were performedwith FlowJo™ (Tree Star, Ashland, Oreg.) software. Stainings wereperformed for: CD3, CD80, and CD95 (Becton Dickinson mAbs 145-2C11,16-10A1, and Jo2, respectively); CD4, CD8a, TCR Vα2, CD19, CD86, CD11b,CD11c, F4/80, and CD69 (BioLegend, San Diego, Calif., mAbs RM4-5,53-6.7, B20.1, 6D5, GL-1, M1/70, N418, BM8, and H1.2F3, respectively);B220, GL7, Ly5.1 (eBioscience, San Diego, Calif., mAbs RA3-6B2, GL-7,and A20, respectively); FITC labeled peanut agglutinin (FITC-PNA) wasobtained from Vector Labs (Burlingame, Calif.); anti-MHC II (NIMR-4)from Southern Biotech (Birmingham, Ala.); and anti-TACI/TNFSF13b (mAb166010) from R&D Systems (Minneapolis, Minn.). NP-APC and NPPE wereprepared by conjugation of NP-Osu (Biosearch Technologies, Novato,Calif.) to allophycocyanin or phycoerythrin (both from Sigma-Aldrich) asdescribed for NP2-BSA above. All isotype control mAbs were purchasedfrom BioLegend.

Other Antibodies and Reagents

The anti-CD180 (RP/14) hybridoma was a kind gift from K. Miyake(University of Tokyo, Tokyo, Japan) and the rat IgG2a isotype control(9D6) hybridoma was a gift from R. Mittler (Emory University, Atlanta,Ga.). To ensure equivalence these mAb were sequentially purified on thesame protein G column and tested for endotoxin by LAL gel-clot assays inGlucaShield™ buffer (Associates of Cape Cod, East Falmouth, Mass.).Samples were rejected if endotoxin levels were above 0.025 EU/mgprotein. mAbs were conjugated to NP as described for NP2-BSA above.Final NP-mAb conjugation ratios ranged from NP6 to NP19 as determined byspectrophotometry. In all inoculations the NP ratios were always higherfor the paired isotype than anti-CD180 to control for any possibleeffects due to TI-2 Ag signaling. Chicken OVA (Sigma-Aldrich) wasconjugated to mAbs as previously described (Weir et al., 1986) with anaverage conjugation ratio of 2 OVA per mAb as determined byelectrophoresis. Amount of conjugate administered refers to the mAbcomponent, i.e., 100 μg OVA-αCD180 contains a total mass of 156 μgOVA-αCD180 due to addition of 56 μg OVA to 100 μg of αCD180. Mice wereinoculated i.v. with a fixed volume of 200 μl in PBS except forimmunizations with Ag in alum, which were administered i.p.Alum-precipitated antigens were prepared with Imject (Thermo FisherScientific) according to the manufacturer's instructions. LPS (L2143)was from Sigma-Aldrich. Synthetic TLR agonists R848, and CpG ODN1585(type A)/ODN1826 (type B) were from InvivoGen (San Diego, Calif.). Whenused, agonists were admixed with the immunogen and administered in the200 μL i.v. bolus.

Immunohistochemistry

8 μM frozen spleen sections obtained from mice immunized 4 or 7 dayspreviously with 100 μg NP-αCD180, NP-isotype, or NP-CGG plus alum werestained with anti-B220-eFluor450 (eBioscience), PNA-FITC (Vector Labs)and NP-PE as previously described (Chappell et al., 2012). Images werecollected on an LSM 510 META™ confocal microscope (Carl Zeiss) with LSM510 (v 4.2) software (Carl Zeiss) using 10× objectives at roomtemperature. Images were processed using ImageJ™ (National Institutes ofHealth) and Photoshop™ (Adobe) software.

Statistical Analyses

Raw data of experimental groups were analyzed either by one-way ANOVAfollowed by Bonferroni's Multiple Comparison Test (GraphPadPrism™software, version 4.0a for Macintosh, San Diego, Calif.) or bytwo-tailed, type two Student's t-test for individual paired columns.Columnar data are represented as mean+/−standard error (SEM). A value ofp<0.05 was considered to be statistically significant and assigned *,while p<0.01 and p<0.001 were assigned ** and ***, respectively.

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Example 2

Six to eight weeks old C57BL/6 mice were immunized with the amounts ofOVA-αCD180 conjugate (i.v.), plus 20 μg of TLR7 agonist R848 (whenindicated), as shown in FIG. 7 , or were left untreated (naïve). At theindicated time points, serum was collected and the specific levels ofIgG (FIG. 7A) and IgM (FIG. 7B) were determined by ELISA. The data showthe average of 3 mice per group+SEM. These data show that as little as17.5 μg of OVA-αCD180 can induce a strong IgG Ab response. However, whena TLR7 agonist (R848) is added, only 1.75 μg of OVA-αCD180 is needed toinduce an Ag-specific response greater than that induced with 17.5 μg ofOVA-αCD180 only. Thus, combinations of the conjugate and a TLR7 agonistprovided synergistic induction of the IgG and IgM antibody responses.

Example 3

Six to eight weeks old C57BL/6 mice were immunized with the amounts ofWNVE-αCD180 conjugate (i.v.) (using same conjugation technique as withOVA), or with live WNV-Texas strain (Tx) (f.p.), as indicated in FIG. 8. As controls, mice were immunized (i.v.) with NP/CD180 conjugates (30μg or 15 μg+R848). At the indicated time points, serum was collected andthe specific titers of IgG (FIG. 8A) and neutralizing titers (FIG. 8B)were determined by ELISA and by a plaque reduction neutralization titer(PRNT) viral neutralization assay adapted for WNV (B cells and antibodyplay critical roles in the immediate defense of disseminated infectionby West Nile encephalitis virus. Diamond M S, Shrestha B, Marri A, MahanD, Engle M. J Virol. 2003 February; 77(4):2578-86), respectively. Thedata are an average of 2 mice per group+SEM. PRNT₅₀ indicates theminimal concentration of serum that reduces the number of plaques by 50%compared to controls. There was no difference between control miceimmunized with 30 μg or 15 μg+R848 of NP/CD180 conjugates (not shown).These data show that mice inoculated with anti-CD180 to which purifiedrecombinant West Nile virus (WNV) envelope (E) protein has been attached(WNVE) develop both WNVE specific IgG Abs (FIG. 8A) and neutralizing Absto WNVE (FIG. 8B).

Example 4

Six to eight weeks old C57BL/6 mice were immunized with the amounts ofWNV E protein/αCD180 conjugate (E/CD180) (same as in Example 3) (i.v.),plus 20 μg R848 or 50 μg CpGB (when indicated) as shown in FIG. 9 , orwere infected with 1000 pfu live WNV-Tx (f.p.). As controls, mice wereimmunized (i.v.) with NP/CD180 conjugates (30 μg or 15 μg+R848 or CpGB).After 28 days, serum was collected and neutralizing titers weredetermined by PRNT assay. The data show the average of 3 mice pergroup+SEM. There was no difference between control mice immunized with30 μg or 15 μg plus R848 or CpGb of NP/CD180 conjugates (not shown).These data clearly show that the WNVE-αCD180 conjugate induces aneutralizing Ab response that is enhanced by the addition of an adjuvantto TLR7 (R848) or TLR9 (CpGB). Mice infected with WNV have a higherneutralizing Ab titer, but then these mice are replicating virus andexposed to virus for a couple of weeks, not just a single injection (aswith the WNVE-αCD180 conjugate injection).

Example 5

Six to eight weeks old C57BL/6 mice were immunized with 15 μg of the WNVE protein/αCD180 conjugate described above (E/CD180) plus 20 μg R848 or50 μg CpGB (i.v.), or infected with 1000pfu live WNV Tex. (f.p.). Ascontrols, mice were immunized (i.v.) with 15 μg NP/CD180 conjugate plus50 μg CpGB. After 32 days (d32), mice were challenged with intracranialinjection of 50 pfu of WNV Tex. (i.c.). Mice were monitored formortality for 21 days. The data show the survival/mortality of 2 miceper group. These data (see Table 1) show that B6 mice immunized withWNVE-CD180 conjugates are protected from death induced by intracranial(i.c.) injection of 50 pfu of WNV Tex. strain (WNV-TX). In contrast, themice immunized with NP/CD180+CpGB as a negative control all died by day6.

TABLE 1 Mice immunized with WNVE-aCD180 are protected from lethal WNVchallenge Survival after i.c. Immunization/Group challenge withWNV-TX 1) 1000 pfu WNV-TX 100% > 21 days (pre-exposed, pos. control) 2)NP-aCD180 + CpGB 0%, all dead by day 7 p.i. (no WNV-TX, neg. control) 3)WNVE-aCD180 + CpGB 100% > 21 days 4) WNVE-aCD180 + R848  50% > 21 days

Example 6

OT-I OVA-specific CD8 T cells were transferred into C57BL/6 mice, whichwere then inoculated with 20 μg of αCD180/OVA conjugate (i.v.), alone ortogether with 50 μg of poly I:C (TLR7 agonist) or CpGB (TLR9 agonist),or with 20 μg of isotype/OVA conjugate as a negative control, or withphosphate buffered saline (PBS). On days 3, 7, and 20, OT-I T cells fromperipheral blood were analyzed by gating on CD8+ cells, and Vα2 andVβ5.1/5.2 The data (not shown) demonstrate that mice inoculated withAg-specific CD8 T cells and then αCD180/OVA (without adjuvant) have anexpanded frequency of Ag-specific T cells 3 days later, and then levelswane. If adjuvant is added, then more CD8 T cells expand and survivelonger (See Table 2, with CpGB as the adjuvant). This demonstrates thatAg-anti-CD180 can activate and expand cytotoxic T cell responses, andcombinations with adjuvant provide even better activation and expansionof the cytotoxic T cell response.

TABLE 2 Mice immunized with OVA-aCD180 with have expanded levels ofAg-specific CD8 T cells % OVA Ag-specific CD8 T Immunization cells oftotal CD8 T on: Group Day 3 Day 7 Day 20 1) PBS 1.8 2.1 2.0 2)OVA-Isotype control 2.6 2.0 2.5 3) OVA-aCD180 6.0 2.3 2.5 4)OVA-aCD180 + CpGB 9.6 3.0 3.5

C57BL/6 mice were inoculated day −1 with 500,000 OVA-specific CD8 Tcells (OT-I Tg T cells) and on day 0 inoculated i.v. with either: 1)PBS; 2) 20 mg of OVA attached to G28-1 isotype control mAb; 3) 20 mg ofOVA attached to αCD180; or 4) 20 mg of OVA attached to αCD180+50 mg CpGBadjuvant. Means are shown for 3 mice/group for each time point. Boldnumbers are statistically significantly different from controls.OVA-αCD180 immunization expands CD8 T cell numbers in vivo. Theexpansion is not long-lasting but can be extended by the addition ofadjuvant.

We claim:
 1. A composition, comprising: (a) an antibody or antibodyfragment that binds CD180; and (b) a heterologous antigen attached tothe antibody or antibody fragment that binds CD180.
 2. The compositionof claim 1, wherein the heterologous antigen is a polysaccharide,carbohydrate, or a glycolipid antigen.
 3. The composition of claim 1,wherein the heterologous antigen is an allergen.
 4. The composition ofclaim 1, wherein the heterologous antigen is a pathogen-specificantigen.
 5. The composition of claim 1, wherein the antibody or antibodyfragment that binds CD180 is a CD180 monoclonal antibody or antibodyfragment.
 6. The composition of claim 5, wherein the CD180 monoclonalantibody or antibody fragment comprises a human or animal CD180 bindingdomain linked to an immunoglobulin constant region (Fc) domain that hasimpaired binding to human or animal Fc receptor FcγRIIb and/or to humanor animal complement proteins.
 7. The composition of claim 1, whereinthe composition further comprises an adjuvant.
 8. The composition ofclaim 7, wherein the adjuvant is a toll-like receptor agonist.
 9. Apharmaceutical composition, comprising: (a) the composition of claim 1;and (b) a pharmaceutically acceptable carrier.
 10. The composition ofclaim 2, wherein the antibody or antibody fragment that binds CD180 is aCD180 monoclonal antibody or antibody fragment.
 11. The composition ofclaim 3, wherein the antibody or antibody fragment that binds CD180 is aCD180 monoclonal antibody or antibody fragment.
 12. The composition ofclaim 4, wherein the antibody or antibody fragment that binds CD180 is aCD180 monoclonal antibody or antibody fragment.
 13. The composition ofclaim 10, wherein the composition further comprises an adjuvant.
 14. Thecomposition of claim 11, wherein the composition further comprises anadjuvant.
 15. The composition of claim 12, wherein the compositionfurther comprises an adjuvant.